Heretofore, recovery in the functions of injured nervous tissues has been considered to be very difficult. However, neural stem cells that retain the ability to grow autonomously and pluripotency have been found in the adult brain in recent years. Based on this finding, regenerative medicine has been studied energetically on the central nervous system as well. Cell therapy for replenishing injured cells has been thought to be closest to practical use as the regenerative medicine using stem cells. In the cell therapy, cells provided by a donor (hereinafter, referred to as donor cells) are cultured, grown, and/or induced to differentiate ex vivo. The cells in an appropriate form are administered into a living subject as a recipient to replenish the injured tissue cells of the recipient.
For brain neurological disease, an attempt has been made on ischemia or trauma models, in which cells having differentiation potency into cells of the nervous system are extracted from tissues, then cultured, and transplanted to an individual. For example, the present inventors have already found that a cell fraction containing cells capable of differentiating into neuron cells is present in bone marrow cells and further demonstrated that transplantation of these cells to rat demyelinated spinal cord models remyelinates the demyelinated axons (Patent Document 1).
For using donor cells in the treatment of disease, particularly, in the treatment of disease having symptoms in the acute phase, such as ischemic brain disease attributed to cerebral infarction, it is important to grow the donor cells rapidly and massively. Various attempts to grow cells ex vivo have been made for improving a cell survival rate and enhancing a growth rate.
For example, media supplemented with various growth-promoting substances are used for improving a cell growth rate. For example, Patent Document 2 discloses that leukocyte inhibitory factors increase a cell growth rate. Patent Document 3 discloses a medium containing recombinant human serum albumin for growing hematopoietic cells. Patent Document 4 diseases a method for culturing mesenchymal stem cells in a medium containing vitamin C and a basic fibroblast cell growth factor. Use of other growth factors (e.g., epithelial cell growth factors, nervous cell growth factors, liver cell growth factors, thrombopoietin, and interleukin) is also known in the art.
Culture substrates that more highly enhance a cell growth rate have also been developed. For example, Patent Document 5 discloses a method for culturing mesenchymal stem cells on basement membrane extracellular matrix.
Moreover, serum is also generally used as a growth-promoting substance. Heretofore, a medium supplemented with approximately 10% to 20% foreign animal serum including fetal bovine serum (FBS) or other cell growth factors has been used widely in stem cell culture. However, the animal cells such as FBS differ in composition from lot to lot and also have the problem of possible contamination with pathogens such as virus or prion.
To cope with such problems, serum-free media have also been developed (see e.g., Patent Documents 2 and 3). However, culture in such a serum-free medium hardly produces growth equivalent to that in a serum-supplemented medium under the present circumstances.
On the other hand, in an attempt to use human serum, human adult serum is used because use of fetal serum is difficult from the ethical standpoint (see e.g., Patent Documents 6 to 8). The advantage of use of the adult serum is that autoserum of an individual from which the donor cells are collected can be used. The use of the autoserum is very preferable from the viewpoint of compatibility and safety.
The disadvantage of use of the adult serum is lower growth-promoting activity than that obtained using, for example, FBS. The adult serum exhibits insufficient cell growth-promoting activity by itself and therefore, inevitably requires further adding FBS (Patent Document 6) or adding other growth factors (Patent Document 7) for obtaining growth equivalent to that obtained using FBS. However, even when effects equivalent to those obtained using FBS are obtained by the addition of growth factors or the like, rapid and massive growth cannot be obtained which is applicable to the treatment of disease in the acute phase as described above.
On the other hand, conventional methods for culturing/growing cells collected from a living subject comprise adding heparin during collection of tissues or cells containing blood components from a donor to avoid blood coagulation (see e.g., Patent Document 9 and Non-Patent Documents 1 and 2). In a typical case (e.g., collection of bone marrow cells for usual bone marrow transplantation), the amount of heparin administered is approximately tens of U/mL (approximately 20 to 40 U/mL) with respect to the volume of the cell solution. For example, Patent Document 8 discloses addition, to a bone marrow fluid, of a heparin/buffer solution containing heparin in the range of approximately 5 to 15 U/mL. Patent Document 2 discloses a method, wherein heparin is further contained in a culture medium.
Heparin is also used as a growth aid, in addition to the application to prevent blood coagulation as described above. For example, Patent Document 4 discloses that heparin has the effect of enhancing the affinity of a basic fibroblast growth factor (bFGF) for its receptor. Moreover, Patent Document 10 discloses a modified form of sulfated glycosaminoglycan containing heparin, as an aid for neural stem cell growth. However, even these methods using heparin cannot yet achieve a sufficiently rapid and massive growth rate under the present circumstances.
Meanwhile, living subjects, when injured, have a mechanism where the injury site is autonomously repaired. Thus, a certain degree of injury can be repaired without leaving functional damage. However, the endogenous repair mechanism is not sufficient for a large degree of injury. In this case, recovery may be delayed, or the injury may be repaired incompletely, leaving functional damage. Biological tissues, particularly, nervous tissues or the like, receiving such injury have conventionally been thought to be very difficult to repair. However, with the recent finding of stem cells having pluripotency, an attempt has been made to replenish injured cells with such stem cells. For example, Patent Document 11 discloses that mesenchymal stem cells were administered to cerebral infarction model rats in the acute phase of the disease (after 3 to 24 hours of induction of ischemia) and consequently produced significant therapeutic effects.
However, an approach remains to be reported, which is effective for recovering lost functions in the subacute phase or later where the injury site is stabilized to some extent due to the passage of time from injury.    Patent Document 1: Pamphlet of WO02/00849A1    Patent Document 2: National Publication of International Patent Application No. 2002-518990    Patent Document 3: Japanese Patent Laid-Open No. 2005-204539    Patent Document 4: Japanese Patent Laid-Open No. 2006-136281    Patent Document 5: Japanese Patent Laid-Open No. 2003-52360    Patent Document 6: Japanese Patent Laid-Open No. 10-179148    Patent Document 7: Japanese Patent Laid-Open No. 2003-235548    Patent Document 8: Japanese Patent Laid-Open No. 2006-55106    Patent Document 9: Pamphlet of WO01/48147A1    Patent Document 10: Japanese Patent Laid-Open No. 2005-218308    Patent Document 11: Pamphlet of WO2005/007176    Non-Patent Document 1: F. Takaku, “Manual of Bone Marrow Transplantation”, first edition, CHUGAI-IGAKUSHA, 1996, p. 86    Non-Patent Document 2: Y. Miura, ed., “Method of Hematopoietic Stem Cell Culture” first impression of revised 2nd edition, CHUGAI-IGAKUSHA, 1989, p. 38